Infection with hepatitis B virus (HBV) is a major cause of liver disease worldwide and affects more than 1 million people in the United States. Hepatitis induced by hepatitis B virus infection is a complex and intricate process involving interactions of multiple host factors with the virus and/or the viral gene products. The HBV X (HBV) gene plays a crucial role in the life cycle and oncogenic potential of HBV. Since virus-host interactions are central to the pathogenesis of viral infection and host injury, this project aims to elucidate the cellular and molecular mechanisms of HBX-host interactions during HBV infection. We have previously shown that HBX interacts with the proteasome complex in vitro and in vivo. The 26S proteasome complex is the predominant cellular factor which degrades cellular proteins in both ubiquitin-dependent and -independent pathways. It has been implicated in the regulation of a variety of transcriptional and cell cycle factors, cellular stress response, and antigen presentation. HBX interacts with the proteasome subunits through a mutually competitive structural relationship. The crucial HBX sequences involved in interaction with the proteasome complex were important for its function as a transcriptional coactivator. We have also shown that HBX functions as a substrate as well as an inhibitor of proteasome. To further study the biological function of HBV, we systematically introduced single amino acid substitutions into the X gene of an infectious WHV clone, and study the biological effects of these X mutants in the woodchuck model. These mutations were targeted to X sequences that have been shown to be important for interaction with the proteasome. Many of the mutants were transactivation negative but none of them were completely replication defective in vitro. In vivo, all the wild-type and some X transfected animals demonstrated evidence of infection with anti-WHc and/or anti-WHs seroconversion. Most of the wild-type and X mutants transfected animals had transient viremia. Some animals were later challenged with infectious WHV. Animals inoculated with X mutants including those with no serologic evidence of infection were protected from the challenge, suggesting prior infection with resulting protective immunity. The inhibition of proteasome function by HBX may account for the multiple actions of HBX and may be an important feature of HBV infection, possibly in helping stabilize viral gene products and suppressing antigen presentation. With our collaborator, our laboratory is also conducting experiments to study the effect of HBX on MHC class I assembly and antigen presentation. Finally, experiments are under way to target the HBX-proteasome interaction as a novel antiviral approach.